Cardiolipin (CL) is a phospholipid which possesses a unique conical shape due to its four hydrophobic tails. In eukaryotes, CL is found only in the inner mitochondrial membrane, where it is associated with the tight curvature and dense folding of the cristae membrane. In the X-linked genetic disease Barth Syndrome (BTHS), disruption of CL remodeling causes a loss of negative curvature and results in reduced cristae density and extreme bioenergetic deficits. Cristae density is critical for function of the electron transport chain (ETC) and the energy-producing oxidative phosphorylation pathway, thus BTHS patients suffer from classical metabolic disorders including cardiomyopathy, growth retardation, and neutropenia. BTHS involves a specific dysfunction in tafazzin, the enzyme responsible for converting monolyso- cardiolipin (MLCL) into mature CL. Since MLCL has only three acyl chains, its shape is less conical than CL and it is expected to impart less negative curvature on the IMM. This proposal explores the possibility of modifying the structure of CL/MLCL by interaction with a small tetrapeptide, SS-31, which is known to selectively target CL in the IMM, where it stabilizes cristae morphology and protects mitochondrial bioenergetics. Because cristae curvature is essential for optimizing the ETC and ATP synthesis, restoration of curvature in BTHS mitochondria by SS-31 may represent a new strategy for rescuing bioenergetic function in the disease. To test this, human- derived BTHS lymphoblasts will be treated with SS-31 and dose-dependent changes to morphology will be measured by electron microscopy (EM). Bioenergetic changes will be measured in parallel by determination of the respiratory control rate and P/O ratio in isolated mitochondria from these cells. CL is largely replaced by MLCL under BTHS, thus the ability of SS-31 to bind MLCL will be confirmed and characterized for the first time through detailed NMR studies. Finally, the ability of SS-31/CL and SS-31/MLCL binding complexes to alter overall membrane curvatures will be determined using liposome fusion assays as well as a recent cristae-like invagination model utilizing giant unilamellar vesicles. Together these studies hope to show cellular proof-of- concept that SS-31 reverts BTHS defects, while testing the novel hypothesis of a curvature-based mechanism of action. Despite the strong correlation between CL curvature and cristae density, CL-targeting drugs have not been thoroughly explored with regards to BTHS, which currently has no specific drug treatment. SS-31 has already shown efficacy in numerous disease states including heart failure, the major cause of BTHS-related death, and is currently undergoing major Phase II trials. Thus this work is poised for rapid translational opportunities should it prove successful. This work may also have direct relevance for other models of heart failure in which SS-31 has shown efficacy but no mechanism has yet been elucidated.